Stable nad/nadh derivatives

ABSTRACT

The present invention provides for stable nicotinamide adenine dinucleotide (NAD/NADH) and nicotinamide adenine dinucleotide phosphate (NADP/NADPH) derivatives of formula (I), enzyme complexes of these derivatives and their use in biochemical detection methods and reagent kits.

PRIORITY CLAIM

The present application is a divisional application based on andclaiming the priority benefit of U.S. patent application Ser. No.12/020,244, filed Jan. 25, 2008, which is a continuation of PCTApplication No. WO 2007/012494, filed Jul. 28, 2006, which claims thepriority benefit of German Patent Application No. DE 10 2005 035 461.0,filed Jul. 28, 2005, each of which are hereby incorporated by referencein their entireties.

TECHNICAL FIELD OF THE INVENTION

The invention concerns stable nicotinamide adenine dinucleotide(NAD/NADH) and nicotinamide adenine dinucleotide phosphate (NADP/NADPH)derivatives, enzyme complexes of these derivatives, and their use inbiochemical detection methods and reagent kits.

BACKGROUND OF THE INVENTION

Measuring systems for biochemical analytics are important components ofclinically relevant analytical methods. This primarily concerns themeasurement of analytes e.g. metabolitcs or substrates which aredetermined directly or indirectly with the aid of an enzyme. Theanalytes are converted with the aid of an enzyme-coenzyme complex andsubsequently quantified. In this process the analyte to be determined isbrought into contact with a suitable enzyme and a coenzyme where theenzyme is usually used in catalytic amounts. The coenzyme is changede.g. oxidized or reduced by the enzymatic reaction. This process can bedetected electrochemically or photometrically either directly or bymeans of a mediator. A calibration provides a direct correlation betweenthe measured value and the concentration of the analyte to bedetermined.

Coenzymes are organic molecules which are covalently or non-covalentlybound to an enzyme and are changed by the conversion of the analyte.Prominent examples of coenzymes are nicotinamide adenine dinucleotide(NAD) and nicotinamide adenine dinucleotide phosphate (NADP) from whichNADH and NADPH respectively are formed by reduction.

Measuring systems known from the prior art are characterized by alimited shelf-life and by special requirements for the environment suchas cooling or dry storage in order to achieve this storage life. Henceerroneous results caused by incorrect, unnoticed, faulty storage cantherefore occur for certain forms of application e.g. in the case oftests which are carried out by the end-users themselves such as glucoseself-monitoring. In particular the exhaustion of desiccants due toopening of the primary packaging for excessive periods can result inmeasuring errors which in some systems can be hardly recognized by theconsumer.

A known measure that can be used to increase the stability ofbiochemical measuring systems is the use of stable enzymes e.g. the useof enzymes from thermophilic organisms. It is also possible to stabilizeenzymes by chemical modification e.g. cross-linking or by mutagenesis.Furthermore, enzyme stabilizers such as trehalose, polyvinylpyrrolidoneand serum albumin can also be added or the enzymes can be enclosed inpolymer networks e.g. by photopolymerization.

It has also been attempted to improve the storage life of biochemicalmeasuring systems by using stable mediators. Thus the specificity oftests is increased and interferences during the reaction are eliminatedby using mediators having the lowest possible redox potential. However,the redox potentials of the enzyme/coenzyme complexes constitute a lowerlimit for the redox potential. If one falls below this limit, thisreaction with the mediators is slowed down or even prevented.

Alternatively it is also possible to use biochemical measuring systemswithout mediators in which for example coenzymes such as the coenzymeNADH are directly detected. However, a disadvantage of such measuringsystems is that coenzymes such as NAD and NADP are unstable.

NAD and NADP are base-labile molecules the degradation paths of whichare described in the literature (N. J. Oppenheimer in The PyridineNucleotide Coenzymes Academic Press. New York, London 1982, J. Everese,B. Anderson, K. You, Editors, chapter 3, pages 56-65). EssentiallyADP-ribose is formed during the degradation of NAD or NADP by cleavageof the glycosyl bonds between the ribose and the pyridine unit. Thereduced forms NADH and NADPH are, however, acid labile: e.g.epimerization is a known degradation path. In both cases the instabilityof NAD/NADP and NADH/NADPH is due to the lability of the glycosyl bondbetween the ribose and the pyridine unit. But even under conditions thatare not drastic such as in aqueous solution, the coenzymes NAD and NADPare already hydrolysed solely by the ambient humidity. This instabilitycan result in inaccuracies when measuring analytes.

A number of NAD/NADP derivatives are described for example in B. M.Anderson in the Pyridine Nucleotide Coenzymes, Academic Press New York,London 1982, J. Everese, B. Anderson. K. You, Editors, Chapter 4.However, most of these derivatives are not accepted well by enzymes. Theonly derivative which has therefore been previously used for diagnostictests is 3-acetylpyridine adenine dinucleotide (acetyl NAD) which wasfirst described in 1956 (N. O. Kaplan, J. Biol. Chem. (1956) 221, 823).This coenzyme is also not accepted well by enzymes and exhibits a changein the redox potential.

The use of other NAD derivatives with a modified pyridine group isdescribed in WO 10/94370. However, modifications of the nicotinamidegroup usually have a direct effect on the catalytic reaction. In mostcases this effect is negative.

In another stabilization concept the ribose unit was modified in orderto influence the stability of the glycosyl bond. This process does notdirectly interfere with the catalytic reaction of the nicotinamidegroup. However, there may be an indirect effect as soon as the enzymebinds strongly and specifically to the ribose unit. In this connectionKaufmann et al. disclose a number of thioribose-NAD derivatives in WO98/33936 and U.S. Pat. No. 5,801,006 and/or WO 01/49247. However, acorrelation between the modification of the nicotinanmide ribose unitand the activity of the derivatives in enzymatic reactions haspreviously not been demonstrated.

CarbaNAD, a derivative without a glycosyl bond was described for thefirst time in 1988 (J. T. Slama, Biochemistry 1989, 27, 183 andBiochemistry 1989, 28, 7866). In this derivative the ribose issubstituted by a carbacyclic sugar unit. Although carbaNAD was describedas a substrate for dehydrogenases, its activity has not yet been provenin clinical biochemical detection methods.

A similar approach was described later by G. M. Blackburn, Chem. Comm.,1996, 2765 in order to synthesize carbaNAD with a methylenebisphosphonate linkage instead of the natural pyrophosphate. Themethylene bisphosphonate shows higher stability towards phosphatases andwas used as an inhibitor for ADP ribosyl cyclase. The aim was not tomake it more resistant to hydrolysis (J. T. Slama, G. M. Blackburn).

Hence the object of the present invention is to provide stablebioanalytical measuring systems for determining an analyte such asglucose which avoid the sensitivity to hydrolysis of NAD/NADP and at thesame time are active as coenzymes in enzyme reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described in more detail below on the basis ofillustrative embodiments. However, the invention is not limited to theillustrative embodiments given here. The illustrative embodiments areshown schematically in the figures. Identical reference numbers in theindividual figures designate elements which are identical or whosefunctions are identical, or which correspond to one another in terms oftheir function.

FIG. 1 illustrates a diagram of an embodiment of a process forsynthesizing carbaNAD (cNAD).

FIG. 2 shows a graph of the results of stressing NAD at 8° C. and 37° C.

FIG. 3 shows a graph of the results of stressing carbaNAD at 8° C. and37° C.

FIG. 4 illustrates a diagram of an embodiment of a process forsynthesizing borano NAD by alkylating ADP, wherein in the case of Y=BH₃,only the beta phosphate of ADP is alkylated.

FIG. 5 illustrates a diagram of an embodiment of a process forsynthesizing pyrrolidinyl NAD (pNAD), including compound numbers andyields of the respective reaction steps being stated next to thestructural formulae.

FIGS. 6A and 6B show absorption spectra of NAD and pNAD.

FIG. 6B shows absorption spectra of NADH and/or pNADH.

FIG. 7 shows fluorescence spectra of NADH and pNADH as a GlucDH complex(emission spectra).

FIG. 8 shows fluorescence spectra of NADH and pNADH as a GlucDH complex(excitation spectra).

FIG. 9 illustrates a comparison of the stability of NAD and pNAD.

FIG. 10A shows absorption spectra of NAD and cNAD.

FIGS. 10B and 10C show absorption spectra of NADH and/or cNADH.

FIG. 11 shows fluorescence spectra of NADH and cNADH as a GlucDHcomplex.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE PRESENT INVENTION

The following description of embodiments of the present invention ismerely exemplary in nature and is in no way intended to limit thepresent invention or its application or uses.

Embodiments of the present invention pertain to a test elementconfigured for determining an analyte, comprising (i) acoenzyme-dependent enzyme or a substrate for such an enzyme and (ii) acompound of the following general formula (I) as the coenzyme:

in which

-   A=adenine or an analogue thereof,-   T=in each case independently denotes O, S,-   U=in each case independently denotes OH, SH, BH₃ ⁻, BCNH₂ ⁻-   V=in each case independently denotes OH or a phosphate group,-   W=COOR, CON(R)₂, COR, CSN(R)₂ in which R in each case independently    denotes H or C₁-C_(z)-alkyl,-   X₁, X₂=in each case independently denote O, CH₂, CHCH₃, C(CH₃)₂, NH,    NCH₃,-   Y=NH, S, O, CH₂,-   Z=a residue comprising a cyclic group with 5 C atoms which    optionally contains a heteroatom selected from O, S and N and    optionally one or more substituents, and a residue CR4₂ wherein CR4₂    is bound to the cyclic group and to X2    -   where R4=in each case independently denotes H, F, Cl, CH₃.        provided that Z and the pyridine residue are not linked by a        glycosidic bond, or a salt or optionally a reduced form thereof.

In one embodiment, W=CONH₂ or COCH₃.

Exemplary substituents on Z are selected from the group consisting ofOH, F, Cl, and C₁-C₂ alkyl which are optionally fluorinated orchlorinated or/and OH-substituted, O—(C₁-C₂-alkyl.

In another embodiment, a first residue V is OH and a second residue V isa phosphate group. Optionally the one OH group and the one phosphategroup can form a ring together with the carbon atoms to which they arebound.

In yet another embodiment, the test element is provided that isconfigured to determine glucose and comprises a glucose dehydrogenaseand a compound of the general formula (I) as mentioned above or a saltthereof.

Surprisingly the compounds according to the invention are generallystable towards hydrolysis and are good substrates in enzymatic detectionmethods and can be used for biochemical diagnostics. This finding is incontrast to that of most of the previously known NAD/NADP derivativessince these derivatives are usually stable for only very short periodsin enzymatic detection methods.

The advantages of the compounds according to the invention compared tothe prior art include:

-   -   high stability,    -   high enzymatic activity,    -   simple and economic synthesis,    -   they can be used in all previously known biochemical detection        methods.

The disadvantages of the previously known biochemical detection methodscan be largely avoided by the provision of stable NAD/NADP derivativesusing the present invention such as in combination with a stabilizingformulation such as for example by enclosing enzymes in polymernetworks. Moreover, it is not necessary to use stabilizing additives.This is particularly advantageous since the lower the number of reactivesubstances involved, the greater is the chance of obtaining a stableformulation for the analyte determination.

Embodiments of the present invention provide test elements comprising anumber of stable NAD/NADP derivatives which have an adequate enzymaticactivity for use as a coenzyme on the test element.

Stable NAD/NADP derivatives can be produced in generally known processesof synthesis. For this the amino group of a cyclic amino alcohol isconverted into a pyridinium derivative by Zincke chemistry. The primaryOH group is subsequently phosphorylated and coupled to an activated AMPderivative to form an NAD derivative. Alternatively the primary OH groupcan also be firstly phosphorylated and then the amino group can beconverted into a pyridine by means of the Zincke reaction.

Another synthetic route is to activate the primary alcohol to form atosylate or iodide and subsequently alkylate ADP.

Embodiments of the test element according to the invention comprise forexample compounds having the following general formula (I′):

in which

-   A=adenine or an analogue thereof,-   T=in each case independently denotes O, S,-   U=in each case independently denotes OH, SH, BH₃, BCNH₂,-   V=in each case independently denotes OH or a phosphate group,-   W=COOR, CON(R)₂, COR, CSN(R)₂ in which R in each case independently    denotes H or C₁-C₂-alkyl,-   X₁, X₂=in each case independently denote O, CH₂, CHCH₃, C(CH₃)₂, NH,    NCH₃,-   Y=NH, S, O, CH₂,-   Z=a saturated or unsaturated carbocyclic or heterocyclic    five-membered ring, in particular a compound of the general formula    (II)

in which a single or double bond can be present between R5′ and R5″,R4=in each case independently denotes H, F, Cl, CH₃,

R5=CR4₂,

if a single bond is present between R5′ and R5″, thenR5′=O, S, NH, NC₁-C₂-alkyl, CR4₂, CHOH, CHOCH₃.R5″=CR4₂, CHOH, CHOCH₃,if a double bond is present between R5′ and R5″, then R5′=R5″=CR4.R6, R6′=in each case independently denote CH, CCH₃or a salt or optionally a reduced form thereof.

Compounds of the following general formula (I″) are another subjectmatter of the invention:

in which

-   A=adenine or an analogue thereof,-   T=in each case independently denotes O, S,-   U=in each case independently denotes OH, SH, BH₃ ⁻, BCNH₂ ⁻,-   V=in each case independently denotes OH or a phosphate group,-   W=COOR, CON(R)₂, COR, CSN(R)₂ in which R in each case independently    denotes H or C₁-C₂-alkyl,-   X₁, X₂=in each case independently denote O, CH₂, CHCH₃, C(CH₃)₂, NH,    NCH₃,-   Y=NH, S, O, OCH₂,-   Z=a saturated or unsaturated carbocyclic or heterocyclic    five-membered ring, in particular compounds of the general formula    (II)

in which a single or double bond can be present between R5′ and R5″,R4=in each case independently denotes H, F, Cl, CH₃.

R5=CR4₂,

if a single bond is present between R5′ and R5″, thenR5′=O, S, NH, NC₁-C₂-alkyl, CR4₂, CHOH, CHOCH₃,R5″=CR4₂, CHOH, CHOCH₃,if a double bond is present between R5′ and R5″, then R5′=R5″=CR4,R6, R6′=in each case independently denote CH, CCH₃provided that if R5=CH₂, T=O, U=in each case denotes OH, V=OH, W=CONH₂,X=O and Y=O then R5′ and R5″ are not simultaneously CHOH,or a salt or optionally a reduced form thereof.

In a preferred embodiment the compounds according to the inventioncontain adenine analogues such as C₈-substituted and N₆-substitutedadenine, deaza variants such as 7-deaza, aza variants such as 8-aza orcombinations such as 7-deaza or 8-aza or carbocyclic analogues such asformycin where the 7-deaza variants can be substituted in the 7 positionwith halogen, C₁-C₆-alkinyl, C₁-C₆-alkenyl or C₁-C₆-alkyl.

In a further embodiment the compounds contain adenosine analogues whichcontain for example 2-methoxydeoxyribose, 2′-fluorodeoxy-ribose,hexitol, altritol or polycyclic analogues such as bicyclic, LNA andtricyclic sugars instead of ribose.

In particular (di)phosphate oxygens can also be isoelectronicallysubstituted such as for example O⁻ by S⁻ and/or by BH₃ ⁻, O by NH, NCH₃and/or by CH₂ and ═O by ═S.

In one embodiment at least one residue IT of the compound according tothe invention is different from OH and in other embodiments at least oneresidue U=BH₃ ⁻.

Yet other embodiments are the derivatives borano carbaNAD, c-pentyl NAD,pyrrolyl NAD, furanyl NAD, carbaNADcyclophosphate, carbaNADP,pyrrolidinyl NAD as well as test elements which contain them:

Biochemical detections of analytes, for example parameters in bodyfluids such as blood, serum, plasma or urine or in samples of wastewater or foods are of major importance in diagnostics. In these teststhe analyte to be determined is brought into contact with a suitableenzyme and a coenzyme provided on a test element.

Hence, another subject matter of the present invention is anenzyme-coenzyme complex comprising a compound according to the inventionin combination with a suitable enzyme.

Any biological or chemical substances that can be detected by a redoxreaction can be determined as analytes e.g. substances which aresubstrates of a coenzyme-dependent enzyme or the coenzyme-dependentenzymes themselves. Examples of analytes are glucose, lactic acid, malicacid, glycerol, alcohol, cholesterol, triglycerides, ascorbic acid,cysteine, glutathione, peptides, urea, ammonium, salicylate, pyruvate,5′-nucleotidase, creatine kinase (CK), lactate dehydrogenase (LDH),carbon dioxide etc.

For the detection of enzyme substrates, embodiments of a test elementmay contain an enzyme that is suitable for detecting the substrate, inaddition to the coenzyme. Suitable enzymes are for exampledehydrogenases selected from glucose dehydrogenase (E.C.1.1.1.47),lactate dehydrogenase (E.C.1.1.1.27, 1.1.1.28), malate dehydrogenase(E.C.1.1.1.37), glycerol dehydrogenase (E.C.1.1.1.6), alcoholdehydrogenase (E.C.1.1.1.1), alpha-hydroxybutyrate dehydrogenase,sorbitol dehydrogenase, and amino acid dehydrogenase e.g. L-amino aciddehydrogenase (E.C.1.4.1.5). Further suitable enzymes are oxidases suchas glucose oxidase (E.C.1.1.3.4) or cholesterol oxidase (E.C.1.1.3.6) oraminotransferases such as aspanate or alanine aminotransferase,5′-nucleotidase or creatine kinase.

For the detection of enzymes, embodiments of a test element may containan enzyme substrate suitable for detecting the enzyme, in addition tothe coenzyme.

Another aspect of the present invention is the use of a compoundaccording to the invention or of an enzyme-coenzyme complex according tothe invention to detect an analyte in a sample by an enzymatic reaction.In this connection the detection of glucose with the aid of glucosedehydrogenase (GlucDH) is an exemplary use.

The change in the coenzyme i.e. in the compound according to theinvention by reaction with the analyte (if the analyte is an enzymesubstrate) or by an analyte-catalysed reaction (if the analyte is anenzyme) can in principle be detected in any desired manner. Basicallyall methods for detecting enzymatic reactions that are known from theprior art can be used. For example, the change in the coenzyme can bedetected by optical methods. Optical detection methods for exampleinclude the measurement of absorption, fluorescence, circular dichroism(CD)), optical rotary dispersion (ORD), refractometry etc. Fluorescencemeasurement in particular is highly sensitive and enables the detectioneven of low concentrations of the analyte in miniaturized systems.

A liquid test can be used to detect an analyte in which the reagent isfor example present in the form of a solution or suspension in anaqueous or non-aqueous liquid or it is present as a powder orlyophilisate. It is, however, also possible to use a dry test, in whichcase the reagent is applied to a carrier, such as a test strip. Thecarrier can for example be a test strip comprising an absorbent or/andswellable material that is wetted by the sample liquid to be examined.

A gel matrix in which an enzyme-coenzyme complex is incorporated can,however, also be used as a detection reagent (cf. DE 1 02 218 45 A 1).

In this case the enzyme can either be polymerized into the matrixtogether with the compound according to the invention or, after thepolymerization, the matrix can be contacted with a solution of thecoenzyme in the presence of the enzyme to form the correspondingenzyme-coenzyme complex.

Another aspect of the present invention concerns a reagent kit and itsuse to detect analytes. The reagent kit can contain a compound accordingto the invention, a suitable enzyme and a suitable reaction buffer.Suitable enzymes have already been described. The reagent kit accordingto the invention can be used in a wide variety of ways and can be usedto determined analytes such as glucose, lactic acid, malic acid,glycerol, alcohol, cholesterol, triglycerides, ascorbic acid, cysteine,glutathione, peptides, urea, ammonium, salicylate, pyruvate,5′-nucleotidase, CK, LDH and carbon dioxide etc. In one embodiment, areagent kit is provided which contains a compound according to theinvention and glucose dehydrogenase (E.C.1.1.1.47) to detect glucose inblood.

The reagent kit according to the invention can be used to detect ananalyte in a dry or liquid test.

Another aspect of the present invention concerns a test strip for thefluorometric or photometric detection of an analyte. Such a test stripcontains a compound as stated above as a coenzyme and a suitable enzymeor an enzyme substrate immobilized on an absorbent or/and swellablematerial. Suitable materials can for example be selected from cellulose,plastics etc.

Another aspect of the present invention comprises a method for detectingan analyte comprising the steps:

-   (a) contacting a sample with a test element or reagent kit according    to the invention comprising a coenzyme; and-   (b) detecting the analyte e.g. on the basis of a change in the    coenzyme.

One aspect of the invention is that the fluorescence emission of thecoenzymes exhibits a bathochromic shift and hence there is lowinterference with the fluorescence emission of other materials of thetest element or/and of the sample.

All embodiments of the aspects of the present invention that aredescribed and/or shown are also intended to apply to other aspects ofthe invention such as, e.g., embodiments of the compounds according tothe invention.

Examples Experimental Preparation of Stable NAD/NADH Derivatives

The preparation of stable NAD/NADH derivatives is shown on the basis ofcarbaNAD (compound 9, FIG. 1) and pyrrolidine NAD (compound 18, FIG. 5)as examples. Additional derivatives can be prepared using appropriateprocesses of synthesis. The corresponding amino alcohols used asstarting reagents are known in the literature:2-amino-1,4-anhydro-2,3-dideoxy-L-threo-pentitol: Huryn, Donna M.;Sluboski, Barbara C.; Tam, Steve Y.; Todaro, Louis J.; Weigele, ManfredTetrahedron letters (1989), 30(46), 6259-62.

3-amino-, (1R,3S)-cyclopentanemethanol, Beres, J.; Sagi, G.; Tomoskodi,L; Gruber, L.; Gulacsi, E.; Otvos, L.; Tetrahedron Letters (1988),29(22), 2681-4.

A) Preparation of carbaNAD I.1R-(−)-exo-cis-5,6-dihydroxy-2-azabicyclo[2.2.1]heptan-3-one (1)

A solution of 16.4 g (147 mmol) 1R-(−)-2-azabicyclo[2.2.I]hept-5-en-3-one in 400 ml acetone is added to a solution of 22.5 g(167 mmol)N-methyl-morpholine-N-oxide in 80 ml deionised water in a 1 Lround-bottomed flask. 15 ml (1.2 mmol) of a 2.5% solution of osiumtetraoxide in tert-butanol is added within 15 min while cooling on ice.The mixture is subsequently stirred overnight at room temperature.

The solvent is removed by distillation in a rotary evaporator. It isstirred with 100 ml and again distilled off in a rotary evaporator.Afterwards it is dissolved in 600 ml deionised water and 35 g activatedcarbon is added. The mixture is stirred vigorously for 1 h and thenfiltered over a Seitz K 250 deep-bed filter. Water is removed from thefiltrate by distillation in a rotary evaporator. The product is usedwithout further purification.

TLC (Merck silica gel 60 F-254): ethyl acetate/methanol/glacial aceticacid 7:2:1 R_(f)0.75 (starting material), 0.53 (1). Staining withTDM/development in a chlorine chamber.

*TDM reagent: Solution 1: 10 gN,N,N′,N′-tetramethyl-4,4′-diamino-diphenyl methane in 40 ml glacialacetic acid and 200 ml deionised water. Solution 2: 20 g potassiumchloride in 400 ml deionised water. Solution 3: Dissolve 0.3 g ninhydrinin 10 ml glacial acetic acid and add 90 ml deionised water. Finishedreagent: A mixture of solution 1 and 2 and addition of 6 ml of solution3.

II.1R-(−)-exo-cis-5,6-dimethylmethylenedioxy-2-azabicyclo[2.2.1]heptan-3-one(2)

The crude product 1 is boiled under reflux for 1 h in 200 ml absoluteethanol. After adding 400 mil (3.26 mol) dimethoxypropane and 250 mg(2.2 mmol) pyridine hydrochloride, the mixture is boiled under refluxfor a further 15 min. After adding 10 ml saturated sodium hydrogencarbonate solution, the solution is evaporated to dryness under a vacuumin a rotary evaporator. 500 ml Chloroform. 150 ml saturated sodiumchloride solution and 75 ml saturated sodium hydrogen carbonate solutionare added to the residue and it is transferred into a separating funnel.After extraction by shaking it is allowed to stand overnight duringwhich phase separation takes place.

The organic phase is separated and the aqueous phase is extracted for afurther two times with 200 ml chloroform in each case. The combinedorganic phases are dried over magnesium sulfate. After removing thedesiccant by filtration, the solvent is removed by distillation underreduced pressure on a rotary evaporator. The crude product (24.9 g=92%)is used without further purification.

TLC (Merck silica gel 60 F-254): ethyl acetate/methanol/glacial aceticacid 7:2:1 R_(f) 0.84. Staining with TDM/development in a chlorinechamber).

III.1R-(−)-4-N-tert-butyloxycarbonyl-exo-cis-5,6-dimethylmethylene-dioxy-2-2-azobicyclo[2.2.1]heptan-3-one(3)

41.5 g (190 mmol) di-tert-butyl dicarbonate and 0.83 g (6.8 mmol)4-dimethyl-aminopyridine are added under argon to a solution of 24.9 g(135.7 mmol) crude product 2 in 450 ml absolute chloroform. The mixtureis boiled under reflux while stirring until the gas evolution ceases.The mixture is filtered over a column that is filled with 40 g silicagel 60 and equilibrated with chloroform. It is washed with 100 mlchloroform. The solvent is removed from the filtrate by distillationunder reduced pressure on a rotary evaporator. The crude product isdried for 60 min at 10 mbar and 40° C. It is used without furtherpurification.

TLC (Merck silica gel 60 F-254): ethyl acetate/hexane 3:2 R_(f)0.85.Staining with TDM/development in a chlorine chamber).

IV.(−)-(1R,2R,3S,4R)-4-(N-tert-butyloxycarbonyl)amino-2,3-dimethyl-methylenedioxy-1-(hydroxymethyl)cyclopentane(4)

The crude product 3 is dissolved at room temperature in 400 mltetrahydrofuran while stirring and 80 ml deionised water is added. Aftercooling to 4° C. 5.3 g sodium borohydride is added all at once andstirred overnight during which the mixture is allowed to slowly heat upto room temperature. 100 ml ethanol is added and it is stirred for 6 hat room temperature. The solvents are removed by distillation underreduced pressure on a rotary evaporator. 300 ml saturated sodiumchloride solution and 650 ml ethyl acetate are added and it istransferred to a separating funnel. The organic phase is separated andthe aqueous phase is again washed with 350 ml ethyl acetate. Thecombined organic phases are dried over magnesium sulfate. After removingthe desiccant by filtration, the solvent is removed by distillationunder reduced pressure on a rotary evaporator. The crude product (42.2g) is purified by means of column chromatography and silica gel 60(column h=93 cm, d=10 cm) eluant THF/hexane 1:3, then THF/hexane 2:3),flow rate 3 L/h. 40 ml fractions are collected. The fractions aremonitored by TLC (Merck silica gel 60 F-254: ethyl acetate/hexane 3:2R_(f) 0.45. Staining with TDM/development in a chlorine chamber). Thesolvent is removed from the combined product fractions by distillationin a vacuum on a rotary evaporator, yield: 24.9 g.

V.(−)-(1R,2R,3S,4R)-4-amino-2,3-dihydroxy-1-(hydroxymethyl)cyclo-pentane(5)

8 ml Deionised water and then 80 ml trifluoroacetic acid are added to11.09 (38.6 mmol) 4. It is stirred vigorously for 6 h at roomtemperature during which a clear pale yellow solution forms. 200 mldeionised water is added and it is evaporated under a vacuum on a rotaryevaporator. 200 ml deionised water is again added and it is againevaporated under a vacuum on a rotary evaporator. The crude product isdissolved in 100 ml deionised water in an ultrasonic bath and filtered.The filtrate is applied to a Dowex 1×8 (100-200 mesh, OH form) ionexchanger column (15×4.9 cm) and eluted with water during which theproduct elutes after about 150 ml in a volume of 300 ml (pH 10.4). Thefractions are monitored by TLC (Merck silica gel 60 F-254:butanol/glacial acetic acid/water 5:2:3 R_(f) 0.42, staining withTDM/development in a chlorine chamber). The solvent is removed from thecombined product fractions by distillation in a vacuum on a rotaryevaporator, yield: 5.2 g colourless oil.

VI. Zincke Salt of the Nicotinamide (6)

58.6 g Dinitrochlorobenzene is melted under nitrogen and then 29.32 gnicotinamide is added to the melt. It is heated for 2.5 h at 110° C. 500ml of a 3:2 (v/v) ethanol/water mixture is added through a reflux coolerand it is boiled under reflux until a solution is formed. After stirringovernight at room temperature, 150 ml 50% ethanol/water and 100 ml waterare added, it is transferred to a separating funnel and washed threetimes with 500 ml chloroform each time. 300 ml and 50 g active carbonare added to the separated aqueous phase which is stirred for 1 h atroom temperature and then filtered over a Seitz K 700 deep-bed filter.The filtrate is concentrated in a vacuum to about 100 ml on a rotaryevaporator during which the bath temperature must not exceed 20° C. Itis diluted with 300 ml water and 70 g sodium tetrafluoroborate is addedat room temperature while stirring. The precipitate is recrystallizedfrom methanol/water. The crystallisate is removed by filtration, washedwith a small amount of acetone and then with diethyl ether and dried for24 h in a high vacuum at 40° C. (yield 21.1 g 23%). The fractions aremonitored by TLC (Merck silica gel 60 F-254: butanol/glacial aceticacid/water 5:2:3 R_(f)=0.56).

VII.(−)-(1R,2R,3S,4R)-4-(3-carboxamidopyridin-1-yl)-2,3-dihydroxy-1-(hydroxymethyl)cyclopentane(6)=carba nicotinamide mononucleoside=carbaNMN

A solution of 4.5 g (31 mmol) cyclopentylamine 5 in 110 ml absolutemethanol is added dropwise within 90 minutes to a solution of 15.3 g(40.7 mmol) of the Zincke salt 6 in 110 ml absolute methanol whilestirring at room temperature. 1 ml diisopropylethylamine is added and itis then stirred for two days at room temperature. 500 ml water is added,transferred into a separating funnel and washed twice with 200 mlmethylene chloride each time. The water is removed from the separatedaqueous phase by distillation under a vacuum on a rotary evaporator. Theresidue is taken up in 100 ml water and purified by columnchromatography on Sephadex C25 (Na+form): column 70×7.5 cm elution ofbuffer A (deionised water) to buffer B 0.35 M NaCl in water, flow rate200) ml/h. 15 ml fractions are collected and monitored by TLC (Mercksilica gel 60 F-254: butanol/glacial acetic acid/water 5:2:3 R_(f)0.22).

The solvent is removed from the combined product fractions bydistillation in a vacuum on a rotary evaporator. The salt-containingresidue is boiled out with 500 ml hot ethanol. It is hot-filtered andallowed to stand for 12 h at room temperature. The precipitate isremoved by filtration and the solvent is removed from the filtrate bydistillation under a vacuum on a rotary evaporator. Yield 7 g.

VII.(−)-(1R,2R,3S,4R)-4-(3-carboxamidopyridin-1-yl)-2,3-dihydroxy-1-phosphatoylmethyl)cyclopentane(6)=carba NMN-monophosphate

A mixture of 20 ml phosphoroxy chloride and 50 ml trimethyl phosphate isadded at 0° C. to a suspension of 7 g (27.7 mmol) carbaNMN in 80 mlanhydrous trimethyl phosphate. It is stirred for 2 h at 0° C. and thenfor 2 h at room temperature. 300 ml water is added while cooling on iceand the mixture is evaporated to 10 ml under a vacuum on a rotaryevaporator. It is taken up in 100 ml water, filtered and purified bymeans of column chromatography on Sephadex C25 (NEt3H+form): column 66×9cm, elution of buffer A (deionised water) to buffer B) 0.60 M ammoniumacetate, flow rate 200 ml/h. 15 ml fractions are collected and monitoredby TLC (Merck silica gel 60 F-254 plates: isobutyric acid/ammmonia/water66:1:33, R_(f)0.25). The solvent is removed from the combined productfractions by distillation in a vacuum on a rotary evaporator. Theresidue is dissolved in 100 ml water and lyophilized. This procedure isrepeated three times. Yield 4.0 g.

IX. carbaNAD (9)

A solution of 1.25 g (30 mmol) AMP morpholidate in 40 ml absolute DMF isadded dropwise within 1 h at room temperature to a mixture of a solutionof 3.31 g (10 mmol) carbaNMN monophosphate in 40 ml absolute DMF and 78ml (39 mmol) 3.5% tetrazole in absolute acetonitrile. The mixture isstirred for 2 days at room temperature.

The pH is adjusted to 6.5 using an aqueous 10% KHCO₃ solution whilecooling on dry ice/acetone. It is diluted with 500 ml water andcarefully concentrated to dryness in a vacuum on a rotary evaporator.The residue is dissolved in 150 ml deionised water and purified bycolumn chromatography on Sephadex QAE 25 (NEt3H+form): column 65×4.5 cm,elution of buffer A (deionised water) to buffer B) 1 M triethylammoniumcarbonate, flow rate 200 ml/h: 15 ml fractions are collected andmonitored by TLC (Merck silica gel 60 F-254 plates: isobutyricacid/ammonia/water 66:1:33, R_(f)0.47).

The solvent is removed by distillation from the combined productfractions by distillation in a vacuum on a rotary evaporator. Theresidue is dissolved in 100 ml water and lyophilized. This procedure isrepeated three times. Yield 1.1 g.

Examination of the Stability of carbaNAD

A 10 mM solution of carbNAD) and/or NAD is stressed at pH 8 in 0.1 Mpotassium phosphate buffer. The content is determined by means of HPLCchromatography after 0.25, 75 and 175 h.

Buffer A: 100 mM KHPO₄+10 mM tetrabutylammonium hydrogen sulfate, pH 6.9buffer B: buffer A+acetonitrile 1:1flow rate 1.0 ml/min detection: 254 nmRP18 column 1, 125 diameter 4.6 mmgradient: in 40 min to 35% buffer B, hold for 2 min and then change to0% buffer A within 3 min.

The HPLC area percentages after stressing for the various times areshown in FIGS. 2 and 3.

The occurrence of decomposition products (nicotinamide, ADP-ribose. AMP.ADP and the unknown decomposition products for NAD and the unknowndecomposition products Y1 and Y2 for cNAD) show that cNAD is very stablecompared to NAD.

B) Preparation of Pyrrolidinyl-NAD

I. Synthesis of pNAD 1st Stage (Compound 10)

Trans-N-t-BOC-O-mesyl-4-hydroxyl-L-prolinol (35.4 g, 120 mmol) wasdissolved in 500 ml DMF and sodium azide (15.6 g, 240 mmol) dissolved in75 ml water was added and heated for 5 h to 70° C. It was subsequentlystirred further overnight at room temperature, the mixture was pouredinto 1000 ml saturated sodium chloride solution and extracted with ethylacetate. The ethyl acetate was dried with Na₂SO₄ and subsequentlyevaporated.

32.8 g (>100%) residue was formed (theoretical value: 29 g).

The crude product was directly processed further after TLC and MSmonitoring. A thin layer chromatography on a KG 60 F-254 plate (mobilesolvent: ethyl acetate/sprayed with ninhydrin) was carried out for themonitoring:

trans-N-t-BOC-O-mesyl-4-hydroxy-L-prolinol R_(f): 0.49

product R_(f): 0.78

MS ESI ES+242

The identity of the product was also confirmed by NMR analysis.

*trans-N-t-BOC-O-mesyl-4-hydroxyl-L-prolinol is commercially availablefrom Sanochenmia Phannazeutika AG, Cat. No. P-719.

II. Synthesis of pNAD 2nd Stage (Compound 11)

Compound 10 (120 mmol) was mixed in 500 ml methanol with 2.0 g Pd-carbon(10%) and hydrogenated for 12 h at 30 mbar. In this process the reactionflask was flushed several times with H₂, the catalyst was removed bysuction filtration and it was concentrated.

A colourless oil was formed (the oil should be immediately processedfurther due to its high air-sensitivity).

MS ESI ES+217 present

TLC (isohexane/ethyl acetate 1/1/KG 254 F/ninhydrin): product remains atthe start.

The identity of the product was also confirmed by NMR analysis.

III. Synthesis of pNAD 3rd Stage (Compound 12)

120 mmol of compound 11 (MW: 216.28) was mixed in 500 ml dioxanecontaining NaHCO₃ (11.0 g, 130 mmol) and Fmoc chloride (33.8 g, 130mmol) and stirred overnight at room temperature. The resulting saltswere removed by filtration, the solution was evaporated and the residuewas purified over a silica gel column (isohexane and isohexane/EE8/2-1/1).

The main fraction yielded 39.0 g=74.1%*(theoretical value=52.6 g).

TLC (KG 60 F254 mobile solvent isohexane/ethyl acetate 2:1): R_(f)0.13

MS ESI ES+439/+339

The identity of the product was also confirmed by NMR analysis.

*Yield refers to the educt of the 1st stage.

IV. Synthesis of pNAD 4th Stage (Compound 13)

Compound 12 from stage 3 (7.08 g, 16.1 mmol) was dissolved in 80 mltrimethyl phosphate and subsequently cooled to 0° C. in an ice bath.POCl₃ mixed with trimethyl phosphate (13 ml freshly distilled POCl₃ in13 ml trimethyl phosphate) was added to a dropping funnel and added inportions within 20 min under argon. The temperature increased in anexothermal reaction to up to 5° C. Subsequently 2.6 ml pyridine wasadded and it was stirred for a further 40 min at 0° C. and under argon.

The reaction solution was carefully added dropwise to 800 ml ice-cooled1 M triethylammonium hydrogen carbonate solution (pH=8). After theaddition was completed, it was stirred for a further 1 h. The slightlyturbid solution was subsequently added (rapidly) dropwise to 1 Lsaturated NaCl solution. It was stirred further overnight to improve thecrystallization. The precipitate was removed by filtration. The residuewas desalted over a Diaion column. For this purpose 500 g Diaion wasadded to isopropanol/water 1/1 and allowed to swell overnight. Diaionwas filled into the column and rinsed with water. A slurry of theresidue was formed in 100 ml water pH 3.5 (acetic acid) which wassubsequently applied to a column and rinsed with water (pH 3.5) until itwas free from sodium chloride. The substance was then eluted from thecolumn with 25% isopropanol (pH 3.5). The solution was evaporated in ahigh vacuum at room temperature.

Residue=2.6 g=31.3%

TLC RP8 F254/MeOH/water 9/1

MS ESI ES−517.13

The identity of the product was also confirmed by NMR analysis.

V. Synthesis of pNAD 5th Stage (Compound 14)

A mixture of compound 13 from stage 4 (4.10 g, 7.9 mmol) in 250 mlmethanol and 83 ml 25% ammonia was stirred overnight at room temperatureand evaporated in a vacuum at room temperature. The residue was taken upin 200 ml water and stirred out three times with 100 ml ethyl acetate.Insoluble components were removed by filtration; the clear water phasewas separated and again evaporated at room temperature.

Residue=2.56 g=100%

MS ESI ES−295

In order to remove the NH₃ cations, the residue was dissolved 2× inHünig's base and again evaporated each time in a high vacuum.

VI. Synthesis of pNAD 6th Stage (Compound 15)

The Zincke salt (2.66 g, 8.99 mmol) was submitted partly dissolved in 50ml methanol and compound 14 from stage 5 (2.56 g, 8.31 nmol) dissolvedin 50 ml methanol was added dropwise while stirring. The mixturecoloured red and slowly dissolved. It was stirred further overnight atroom temperature and the precipitate was removed by filtration. Thefiltrate was evaporated in a vacuum, taken up in 100 ml water andextracted three times with ethyl acetate.

The ethyl acetate phase contains the by-product dinitroaniline, thewater phase contains the product and the remaining Zincke salt. Thewater phase was evaporated in a vacuum at room temperature and 10 mlwater was added to the residue that was obtained, which was stirred for10 min on a magnetic stirrer and insoluble components were removed byfiltration. The product remained dissolved. This solution was applied toa Diaion HP20 column (1000 mil) that had been rinsed with water and wasrinsed two times with 1000 ml water. Subsequently it was rinsed withwater/5% isopropanol and positive fractions (detected by TLC RP8 MeOH/W9/1) were evaporated at room temperature. The residue was trituratedwith isopropanol and suction filtered with the aid of diethyl ether.

Residue=1.6 g=47.9%

TLC RP8 254 MeOH/W 9/1

MS ES−400.1/ES+402.0 also exhibits the double mass

The identity of the product was also confirmed by NMR analysis.

VIIa. Synthesis of pNAD Stage 7a (Compound 16)

A mixture of AMP acid (adenosine monophosphate-free acid) (10 g, 27.5mmol) in 60 ml methanol (dried with sodium) and 5.3 ml (60 mmol)morpholine (freshly distilled) was stirred until a clear solution wasformed. Subsequently 17 g (82.5 mmol) N,N′-dicyclohexyl carbodiimide(DCC) was added and stirred overnight at room temperature whileexcluding moisture. The precipitate (DCH) that was formed was suctionfiltered and the filtrate was rotary evaporated at 30° C. Subsequentlyit was stirred out with 150 ml H₂O/150 ml diethyl ether and againfiltered. After phase separation the aqueous phase was again extractedtwice with 75 ml diethyl ether each time. The aqueous phase wassubsequently rotary evaporated at room temperature. The residue wasdissolved a further two times in pyridine and each time again rotaryevaporated in a high vacuum.

VII. Synthesis of pNAD 7th Stage (Compound 17)

A mixture of AMP morpholidate (compound 16 from stage 7a) (2.53 g, 3.61mmol), compound 15 from stage 6 (1.60 g. 3.98 mmol), MnCl₂ solution informamide 0.2 M*(27.1 ml, 5.42 mmol) and anhydrous MgSO₄ (0.87 g, 7.95mmol) were stirred overnight at room temperature and after this timewere largely converted as determined by TLC (RP8 MeOH/W 9/1). Thereaction mixture was precipitated with acetonitrile and suctionfiltered.

Residue=5.3 g (theoretical yield 2.64 g)**

MS ESI ES−729.3=product, ES−415=cation of AMP morpholidate,ES−400.2/ES+402.1 residues of compound 15 (stage 6)

TLC RP 8 F254 Rf0.085

*In order to prepare this solution 2516 mg anhydrous MnCl₂ was dissolvedin 100 ml 99.99% formamide while stirring and subsequently 4A molecularsieve was added.

**The residue was further processed as a crude product.

VIII. Synthesis of pNAD 8th Stage (Compound 18, Pyrrolidinyl-NAD)

5.0 ml trifluoroacetic acid (TFA) was added to 500 mg compound 17 fromstage 7 (crude product, contains about 50% salts) and stirred for 1 h atroom temperature and subsequently concentrated by evaporation. 500 milcolourless oil was formed as the residue MS ESI ES−729.24 (addition ofNH₃ necessary)

2 Portions of 200) mg and 300 mg were each purified in 2 separationsteps:

First Separation Step:

Fractogel EMD SO3-s column: D (inner)=14 mm L (packing)=85 mm

I. Conditioning

(flow rate 5 ml/min)

-   -   a) 100 ml H₂O    -   b) 200 ml 0.25 M H₂SO⁴    -   c) 100 ml H₂O    -   d) 200 ml 1 M ammonia solution    -   e) 100 ml H₂O

II. Separation:

-   -   a) apply 200 ml substance dissolved in 5 ml H₂O    -   b) elute with a gradient of H₂O→0.2 M NH₄HCO₃        -   solution. (mobile solvent A=250 ml H₂O submitted    -   in an Erlenmeyer flask and stirred on a magnetic stirrer,        -   pumped onto the column at a rate of 5 ml/min mobile solvent            B=0.2 M NH₄HCO₃ solution pumped at 2.5 ml/min to A).

III. Fractionation:

-   -   a) fractions each of 3 ml    -   b) 1st peak impurities    -   c) 2nd peak after about 70 ml preliminary eluate=substance

IV. Reconditioning:

-   -   a) 100 ml 1 M ammonium solution    -   b) 100 ml H₂O

2nd Separation Step:

Diaion HP20, column D (inner)=30 mm L (packing) 130 mm eluted with 100ml H₂O and 100 ml H₂O/5% isopropanol.

The substance already elutes with the water phase; only impurities arepresent in the isopropanol fraction.

3 fractions were obtained according to analytical HPLC:

-   -   F1=13.5 mg    -   F2=5.5 mg    -   F3=11.5 mg    -   Total=30.5 mg=12.2%

The identity of the pyrrolidinyl NAD) (compound 18) was confirmed by NMRanalyses.

Glucose Dehydrogenase Assay for pNAD

In order to examine the role of pNAD as a cofactor for glucosedehydrogenase (GlucDH), a glucoseDH activity assay in 0.1 M iris/0.2 MNaCl (pH 8.5) buffer was carried out. The concentration of glucose was80 mM. pNAD and NAD concentrations of 0.05-0.5 mM were used. To this, 10mg/ml (pNAD) or 0.002 mg/ml (NAD) [83 or 0.017 μM respectively] GlucDHwas added. The assay was carried out at room temperature and theenzymatic reaction was monitored by recording absorption spectra atregular time intervals. The values shown in table 1 refer to anabsorption measurement after 4 min.

TABLE 1 (p)NAD (mM) U/ml % activity [GlucDH] used 0.05 NAD 539 100  0.02mg/ml 0.4 NAD 1556 100 0.002 mg/ml 0.05 pNAD 0.00017 0.00003 10 μL 10mg/ml 0.4 pNAD 0.0024 0.00015 10 μL 10 mg/mlAbsorption Spectra of pNAD and pNADH

Absorption spectra of NAD and pNAD and/or NADH and pNADH are shown inFIGS. 6A and 6B. NAD and pNAD exhibit an absorption maximum at 260 nm.pNADH i.e. pNAD after the GlucDH activity assay exhibits a red shift ofthe absorption maximum by about 10 nm (FIG. 6B) compared to NADH.

Fluorescence spectra of NADH and pNADH as GlucDH complexes areadditionally shown in FIGS. 7 and 8. The spectra were in each caserecorded after the GlucDH activity assay. FIG. 7 shows emission spectraof NADH/pNADH-GlucDH complexes at excitation wavelengths of 340 and 370nm. The emission spectra of NADH and pNADH at 370 am excitationwavelength are similar. FIG. 8 shows an excitation spectrum for anNADH/pNADH-GlucDH complex at an emission wavelength of 460 nm. pNADHexhibits a broader excitation spectrum than NADH. The spectra were alsorecorded after GlucDH activity assays.

Investigation of the Stability of pNAD

In order to examine the stability of pNAD compared to NAD, the sameamounts of NAD and pNAD were each taken up in 0.15 M KPO₄. 1 M NaClbuffer (pH 7.0) and incubated at 50° C. The decomposition of NAD and/orpNAD were monitored by HPLC. FIG. 9 shows the percentage areas of the(p)NAD amounts compared to the (p)NAD amounts at time zero. The figureshows that pNAD is very stable compared to NAD.

C) Preparation of carbaNAD Cyclophosphate (19)

79 mg (0.1 mmol)05′-(hydroxy-morpholino-phosphoryl)-O₂′,O₃′-hydroxy-phosphoryl-adenosine,N-cyclohexyl-morpholine-4-carbonimidic acid cyclohexylamine saltdihydrate (dried by coevaporation with pyridine (Morphat et al., J. Am.Chem. Soc. 83; 1961; 663-675), 44 mg (0.105 mmol) carbaNMN monophosphateand subsequently 25 mg dry magnesium sulfate were added to 0.74 ml of a0.2 manganese chloride solution in absolute formamide. The mixture wasstirred under argon for three days in a closed reaction vessel andsubsequently added to 10 ml acetonitrile while stirring. The precipitatewas removed by filtration, purified by RP chromatography on a RP 18Hypersil ODS, 250×21.2 mM, 5 μm column using a 0% B to 100% B gradientfor 60 min: Eluant A: 0.1 M triethylammonium acetate, eluant B: 1:4mixture of 0.1 M triethylammonium acetate and acetonitrile, flow rate:10 ml/min. The elution was monitored by detection at 260 nm. The mainfraction was collected and lyophilized 5 times in order to remove thetriethylammonium acetate. The triethylammonium salt was converted intothe free acid with Dowex 50 WX2 and subsequently into the lithium salt.Yield: 10 mg.

D) Preparation of carbaNADP (20)

Three times four units ribonuclease T2 were added within 6 h at 37° C.to a solution of 2.2 mg carbaNAD cyclophosphate lithium salt (19) in 1ml Bis-tris-propane buffer (0.02 M, pH 7.5). The mixture was keptovernight at 37° C. The enzyme was denatured by heating to 65° (C for 20min. After filtration a purification was carried out by RPchromatography on a RP 18 Hypersil ODS, 250×21.2 mm, 5 μm column using agradient of 0% B to 100% B for 60 min. Eluant A; 0.1 M triethylammoniumacetate; eluant B: 1:4 mixture of 0.1 ml triethylammonium acetate andacetonitrile; flow rate: 10 ml/min. The elution was monitored bydetection at 260 nm. The main fraction was collected and lyophilized 5times in order to remove the triethyl-ammonium acetate.

Mass spectrum (MALDI Applied Biosystems Voyager System 6327: calculated742.45. found: 743.17).

E) Glucose Dehydrogenase Activity Assay for cNAD

A glucose dehydrogenase activity assay for cNAD compared to NAD wascarried out as described under 1) for pNAD. For this purpose glucosedehydrogenase concentrations of 0.1 (cNAD) and 0.002 mg/ml (NAD) [0.83and 0.017 μM respectively] were used. The amounts used and the resultsare shown in table 2.

TABLE 2 (c)NAD (mM) U/ml % activity [GlucDH] used 0.05 NAD 430 100 0.002mg/ml 0.1 NAD 556 100 0.002 mg/ml 0.05 cNAD 2.7 0.63 0.1 mg/ml 0.1 cNAD5.3 0.95 0.1 mg/mlF) Absorption Spectra of cNAD and CNADH

FIGS. 10A, 10B and 10C show absorption spectra of NAD and cNAD. NAD aswell as cNAD have an absorption maximum at 260 nm. FIG. 10B showsabsorption spectra of NADH and cNADH where the spectra were in each caserecorded after a glucose dehydrogenase activity assay. The absorptionmaximum of cNADH exhibits a red shift of 20 nm. Further absorptionspectra for NADH and cNADH are shown in FIG. 10C in which differentconditions for the associated glucose dehydrogenase activity assay wereselected as stated in the legends.

FIG. 11 additionally shows fluorescence spectra of NADH and cNADH asGlucDH complexes. The spectra were recorded at an excitation wavelengthof 370 nm after a glucose dehydrogenase activity assay. NADH as well ascNADH exhibit an increase of the fluorescence signal when treated withGlucDH.

The features disclosed in the above description, the claims and thedrawings may be important both individually and in any combination withone another for implementing the present invention in its variousembodiments.

It is noted that terms like “preferably”, “commonly”, and “typically”are not utilized herein to limit the scope of the claimed invention orto imply that certain features are critical, essential, or evenimportant to the structure or function of the claimed invention. Rather,these terms are merely intended to highlight alternative or additionalfeatures that may or may not be utilized in a particular embodiment ofthe present invention.

For the purposes of describing and defining the present invention it isnoted that the term “substantially” is utilized herein to represent theinherent degree of uncertainty that may be attributed to anyquantitative comparison, value, measurement, or other representation.The term “substantially” is also utilized herein to represent the degreeby which a quantitative representation may vary from a stated referencewithout resulting in a change in the basic function of the subjectmatter at issue.

Having described the present invention in detail and by reference tospecific embodiments thereof, it will be apparent that modification andvariations are possible without departing from the scope of the presentinvention defined in the appended claims. More specifically, althoughsome aspects of the present invention are identified herein as preferredor particularly advantageous, it is contemplated that the presentinvention is not necessarily limited to these preferred aspects of thepresent invention.

1. A method for detecting an analyte in a fluid sample comprising thesteps of (a) providing a test element, the test element carrying anenzyme and a compound of the general formula (I″);

in which A=adenine or an analogue thereof, T=in each case independentlydenotes O, S, U=in each case independently denotes OH, SH, BH₃ ⁻, BCNH₂⁻, V=in each case independently denotes OH or a phosphate group, W=COOR,CON(R)₂, COR, CSN(R)₂ in which R in each case independently denotes H orC₁-C₂-alkyl, X₁, X₂=in each case independently denote O, or NH, Y=NH, S,O, CH₂ Z=a saturated or unsaturated carbocyclic or heterocyclicfive-membered ring of the general formula (II)

in which a single or double bond can be present between R5′ and R5″,R4=in each case independently denotes H, F, Cl, and CH₃, R5=CR4₂, if asingle bond is present between R5′ and R5″, then R5′=O, S, NH,NC₁-C₂-alkyl, CR4₂, CHOH, CHOCH₃, R5″=CR4₂, CHOH, CHOCH₃ if a doublebond is present between R5′ and R5″, then R5′=R5″=CR4, R6, R6′=in eachcase independently denote CH, CCH₃ provided that if R5=CH₂, T=O, U=ineach case OH, V=OH, W=CONH₂, X₁, X₂=O and Y=O, then R5′ and R5″ are notsimultaneously CHOH, or a salt or optionally a reduced from thereof; and(b) analyzing an enzymatic reaction involving the analyte andcorrelating a change in the compound or the enzyme to a quantity of theanalyte.
 2. A method of determining an analyte comprising the steps of:(a) providing a test element configured for determining an analyte,comprising (i) a coenzyme-dependent enzyme or a substrate for such anenzyme and (ii) a compound of the following general formula (I) as thecoenzyme:

in which A=adenine or an analogue thereof, T=in each case independentlydenotes O, S, U=in each case independently denotes OH, SH, BH₃ ⁻, BCNH₂⁻, V=in each case independently denotes OH or a phosphate group, W=COOR,CON(R)₂, COR, CSN(R)₂ in which R in each case independently denotes H orC₁-C₂-alkyl, X₁, X₂=in each case independently denote O, CH₂, CHCH₃,C(CH₃)₂, NH, NCH₃, Y=NH, S, O, CH₂ Z a residue comprising a cyclic groupwith 5 C atoms which optionally contains a heretoatom selected from O, Sand N and optionally one or more substituents, and a residue CR4₂wherein CR4₂ is bound to the cyclic group and to X₂, where R4—in eachcase independently denotes H, F, Cl, CH₃, provided that Z and thepyridine residue are not linked by a glycosidic bond, or a salt oroptionally a reduced form thereof, (b) contacting the test element witha sample; (c) measuring a value relating to a change in one of theenzyme and coenzyme; and (d) correlating the value to a concentration ofthe analyte, wherein the analyte is at least one of the analytesselected from the group consisting of glucose, lactic acid, malic acid,glycerol, alcohol, cholesterol, triglycerides, ascorbic acid, cysteine,glutathione, peptides, urea, ammonium, salicylate, pyruvate,5′-nucleotidase, creatine kinase (CK), lactate dehydrogenase (LDH) andcarbon dioxide.
 3. A method for detecting an analyte comprising thesteps of: (a) contacting a sample with a reagent kit comprising incombination a suitable enzyme, a suitable reaction buffer, and acompound of the general formula (I″):

in which A=adenine or an analogue thereof, T=in each case independentlydenotes O, S, U=in each case independently denotes OH, SH, BH₃ ⁻, BCNH₂⁻, V=in each case independently denotes OH or a phosphate group, W=COOR,CON(R)₂, COR, CSN(R)₂ in which R in each case independently denotes H orC₁-C₂-alkyl, X₁, X₂=in each case independently denote O, or NH, Y=NH, S,O, CH₂ Z=a saturated or unsaturated carbocyclic or heterocyclicfive-membered ring of the general formula (II)

in which a single or double bond can be present between R5′ and R5″,R4=in each case independently denotes H, F, Cl, and CH₃, R5=CR4₂, if asingle bond is present between R5′ and R5″, then R5′=O, S, NH,NC₁-C₂-alkyl, CR4₂, CHOH, CHOCH₃, R5″=CR4₂, CHOH, CHOCH₃ if a doublebond is present between R5′ and R5″, then R5′=R5″=CR4, R6, R6′=in eachcase independently denote CH, CCH₃ provided that if R5=CH₂, T=O, U=ineach case OH, V=OH, W=CONH₂, X₁, X₂=O and Y=O, then R5′ and R5″ are notsimultaneously CHOH, or a salt or optionally a reduced from thereof, and(b) detecting the analyte.
 4. The method according to claim 3, whereinthe analyte is detected photometrically or fluorometrically.